The long-term objective of this proposal is to generate novel immunogens that are capable of eliciting potent HIV-1 neutralizing antibodies. The entry of HIV-1 into target cells is initiated by the binding of viral envelope glycoproteins (Envs) to the CD4 receptor and CCR5 or CXCR4 coreceptors. The highly conserved coreceptor binding sites are induced following gp120-CD4 binding and overlapped with so-called CD4-induced (CD4i) epitopes. Anti-CD4i antibodies (Abs) can be elicited by Envs stabilized in CD4- bound conformation, but these Abs have very limited neutralization activity against primary HIV-1 isolates. Our preliminary results and recent findings by other groups demonstrate that the CD4i epitopes are likely to be distinguishable from the exact coreceptor binding sites. On the other hand, highly unusual CD4-independent HIV-1 viruses are able to infect cells lacking CD4;therefore, the coreceptor binding sites are stably exposed on the Envs. Zhang et al. reported that sgp140 Envs from a CD4-independent HIV-1 isolate R2 elicited extensively cross-reactive anti-HIV-1 neutralizing Abs, which suggested that the coreceptor binding sites stably exposed on the CD4-independent Envs might be able to elicit a novel subset of Abs that are capable of broadly neutralizing primary HIV isolates. However, it is not clear whether the capability of eliciting extensively cross-reactive neutralizing Abs is a property of the specific R2 Envs or a property of the CD4-independent Envs. Kolchinsky et al. generated a CD4-independent CCR5-tropic HIV-1 isolate, ADA/HX N197S, which contains a point mutation in gp120 (N197S) and chimeric sequences in gp41 with an ADA sequence from N-terminal to a.a. 613 and an HXB2 sequence thereafter. In this proposal, we will use three ADA-based Envs that express increasing degrees of CD4-independence (native ADA, ADA N197S, and ADA/HX N197S) to evaluate whether exposure of the coreceptor binding sites on the CD4-independent Envs plays an important role in eliciting improved anti-HIV-1 neutralizing Ab. The studies in this proposal are expected to provide a proof-of-concept evaluation of our hypothesis that exposure of the coreceptor binding sites on CD4-independent Envs will induce improved antibody responses to neutralize HIV-1 infection. PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to develop a safe and effective preventive vaccine against HIV/AIDS. The applicant will evaluate if an HIV envelope glycoprotein that is able to mediate CD4-independent entry is capable of inducing an improved immune response against HIV. The completion of this application will have great impact on design of an effective HIV/AIDS vaccine.